Biomolecules, Metabolism and Bioenergetics

DNA structure and base composition

Double-stranded DNA consists of two antiparallel strands held together by hydrogen bonds between complementary bases. Adenine pairs with thymine through two hydrogen bonds, and guanine pairs with cytosine through three. Chargaff's rules follow directly from this pairing: in double-stranded DNA, [A] = [T] and [G] = [C], so purines and pyrimidines are equal in total. The G+C content is therefore 100 - (A+T), and since A+T = 2[A], knowing only the adenine percentage fixes the whole base composition. Because G–C pairs carry three hydrogen bonds, GC-rich DNA has a higher melting temperature. For example, if adenine is 20%, then thymine is also 20%, so G+C = 60%.

Length and mass of DNA: the rise per base pair in B-DNA is about 0.34 nm, so the contour length of a duplex equals the number of base pairs multiplied by 0.34 nm. The mass of a molecule is the number of base pairs multiplied by the mass of one base pair. Lengths given in different units must be converted to a common unit before calculation, since unit conversion is a frequent source of error in this type of problem.

Nucleoside and nucleotide: a nucleoside contains only a nitrogenous base and a pentose sugar. A nucleotide is a nucleoside carrying one or more phosphate groups. Carbon, nitrogen and oxygen occur in both the sugar and the base of a nucleoside, but phosphorus occurs only in the phosphate group. Phosphorus is therefore the element present in nucleotides and absent in nucleosides.

• nucleoside — base plus pentose, with no phosphate. e.g. adenosine
• nucleotide — nucleoside plus one or more phosphates. e.g. AMP, dAMP
• Chargaff's rules — [A] = [T] and [G] = [C] in double-stranded DNA; they do not necessarily hold for single-stranded genomes.

Coupled reaction free energy

Free energy is a state function, so the standard free energy changes of sequential reactions add when the reactions are summed, and any common intermediate cancels. An endergonic step with \Delta G^\circ{}' > 0 can be driven by an exergonic step such as ATP hydrolysis, which has \Delta G^\circ{}' \approx -30 kJ mol^{-1}, provided the net \Delta G is negative. Adding the reaction for ATP hydrolysis to the reverse of a second reaction gives the free energy of the coupled process.

The same additivity applies to equilibrium constants in multiplicative form. Since \Delta G^\circ = -RT\ln K, the net constant is the product of the individual constants, K_{net} = K_1 K_2, and \Delta G^\circ_{net} = -RT\ln(K_1K_2) = \Delta G^\circ_1 + \Delta G^\circ_2. Use R = 8.314 J mol^{-1}K^{-1} and convert to kJ mol^{-1} at the end.

Sign and feasibility: a large negative \Delta G corresponds to a very large equilibrium constant. For example, \Delta G = -100 kJ mol^{-1} near 298 K gives K of order 10^{17}, so the equilibrium lies almost completely on the product side and the reaction is effectively irreversible. Thermodynamics, however, governs only feasibility and the position of equilibrium. The rate depends on kinetic factors such as activation energy and enzyme concentration, so the rate of a reaction cannot be predicted from \Delta G alone.

• state function — a quantity that depends only on the initial and final states, so \Delta G values can be added along a pathway.
• energy coupling — driving an unfavourable reaction by linking it to a favourable one, typically through a common intermediate.
• thermodynamics vs kinetics — \Delta G says whether a reaction can proceed spontaneously; it does not say how fast it proceeds.

Apoprotein and prosthetic groups

A prosthetic group is a non-amino-acid component that is tightly or covalently bound to a protein and is required for its function. Examples include heme in hemoglobin, FAD in succinate dehydrogenase and biotin in carboxylases. The protein portion without its prosthetic group is called the apoprotein. The complete functional protein, including its prosthetic group, is called the holoprotein, and a conjugated protein is the same complete species viewed as a protein with a non-protein part. Removing the prosthetic group usually abolishes activity, and activity can be restored by adding the group back.

Leghemoglobin is a heme-containing oxygen-binding protein found in the root nodules of leguminous plants. Nitrogenase is irreversibly inactivated by oxygen, so leghemoglobin keeps the free oxygen concentration low enough to protect the enzyme. It still delivers enough oxygen to support the respiration that supplies ATP for nitrogen fixation.

• apoprotein — protein moiety alone, without its prosthetic group.
• holoprotein — the complete, active protein including its prosthetic group.
• hemoprotein — a protein whose prosthetic group is heme, so it is not an apoprotein.
• lipoprotein — a complex of protein with lipid, defined by its lipid content rather than by a missing prosthetic group.

NMR spectroscopy principles

A nucleus gives an NMR signal only if its nuclear spin quantum number I is non-zero. Nuclei with an odd mass number, or with both odd proton and odd neutron numbers, have I \neq 0. The proton (^1H) and carbon-13 (^{13}C) nuclei both have I = 1/2 and are NMR active. The oxygen-16 (^{16}O) and sulfur-32 (^{32}S) nuclei have even numbers of protons and neutrons, so I = 0 and they are NMR inactive. The natural abundance of ^{13}C is only about 1%, so isotopic enrichment is often needed for biomolecular work.

In structural biology, NMR determines three-dimensional structure in solution. Nuclear Overhauser effects give inter-proton distance restraints, and scalar couplings give torsion-angle restraints. Together these restraints allow calculation of the tertiary fold of a protein, including the arrangement of \alpha-helices and \beta-strands.

• nuclear spin — the intrinsic angular momentum of a nucleus; I = 0 means no NMR signal.
• NOE — through-space dipolar effect that reports on proton–proton distances up to about 5 Å.
• MS vs NMR — mass spectrometry measures mass-to-charge ratio, while NMR reports on spatial arrangement.

Protein structural levels

Protein structure is described at four levels. The primary structure is the amino acid sequence. Secondary structure is the local, regularly repeating conformation of the backbone, such as the \alpha-helix and \beta-sheet, stabilised by hydrogen bonds between backbone C=O and N–H groups.

Tertiary structure is the complete three-dimensional fold of a single polypeptide chain. It includes side-chain interactions such as hydrophobic packing, ionic bonds, hydrogen bonds between side chains and disulfide bridges, so it is not restricted to backbone atoms. Quaternary structure describes the assembly of two or more polypeptide subunits into a functional complex, as in the \alpha_2\beta_2 arrangement of hemoglobin.

Methods for secondary structure: circular dichroism in the far-UV region and FTIR spectroscopy of the amide I band report the fractions of helix, sheet and random coil. X-ray crystallography provides atomic coordinates from which these elements can be assigned. Mass spectrometry measures mass-to-charge ratios and gives mass and sequence information but no conformational data, so it cannot determine secondary structure content.

• primary structure — covalent amino acid sequence.
• secondary structure — backbone-level folding, such as \alpha-helix and \beta-sheet.
• tertiary structure — overall fold of one chain, including side-chain interactions.
• quaternary structure — association of multiple chains.

Central carbon metabolism

The tricarboxylic acid (TCA, Krebs) cycle oxidises acetyl-CoA to CO_2 and transfers energy to NADH, FADH_2 and GTP, so it is a catabolic pathway. Several of its intermediates are also withdrawn for biosynthesis: oxaloacetate supplies aspartate and gluconeogenesis, \alpha-ketoglutarate supplies glutamate, and succinyl-CoA supplies heme. A pathway that serves both breakdown and biosynthesis is called amphibolic. Intermediates removed for biosynthesis must be replenished by anaplerotic reactions.

Glycolysis in erythrocytes lacks mitochondria, so glycolysis is the only ATP source and its regulation is important. The main control point is phosphofructokinase-1 (PFK-1). AMP is an allosteric activator of PFK-1 and signals low energy charge, so it stimulates glycolysis. Citrate is an allosteric inhibitor of PFK-1. Fructose 1,6-bisphosphate activates pyruvate kinase in a feed-forward manner. Glucose 6-phosphate inhibits hexokinase by product feedback at the entry step, a control point distinct from PFK-1 and pyruvate kinase.

• amphibolic — serving both catabolic and anabolic functions.
• anaplerotic reaction — a reaction that replenishes TCA cycle intermediates.
• feed-forward activation — an upstream metabolite activates a downstream enzyme, as fructose 1,6-bisphosphate does for pyruvate kinase.

Degree of reduction

The degree of reduction (reductance) of a compound is the number of electron equivalents it releases when it is completely oxidised to CO_2, H_2O, and NH_3. Per carbon mole, for a compound C_cH_hO_oN_n, the Roels expression is \gamma = (4c + h - 2o - 3n)/c. Glucose gives \gamma = 4 per carbon mole, and oxalic acid, C_2H_2O_4, gives \gamma = (8+2-8)/2 = 1. Each carbon in oxalate passes from +3 to +4 on oxidation to CO_2, releasing one electron per carbon atom.

An electron equivalent (e-eq) is one mole of electrons. For acetate, the complete oxidation is \text{CH}_3\text{COO}^- + 2\text{H}_2\text{O} \rightarrow 2\text{CO}_2 + 7\text{H}^+ + 8e^-, so each mole of acetate gives 8 e-eq. To convert a mass concentration to e-eq L^{-1}, divide by the molar mass to get mol L^{-1}, then multiply by the number of e-eq per mole. For example, 10 g L^{-1} of acetate (molar mass 59 g mol^{-1}) gives 8 \times 10/59 \approx 1.36 e-eq L^{-1}.

• e-eq — one mole of electrons, used as a unit of reducing power.
• Roels convention — the standard method of computing \gamma per carbon mole.
• complete oxidation — conversion of carbon to CO_2, which defines the reference state for \gamma.

Chirality and optical activity

A molecule is chiral if it is not superimposable on its mirror image. Optical activity, the rotation of plane-polarised light, requires chirality. Most chiral molecules contain at least one stereogenic carbon bonded to four different groups. Sodium chloride is ionic and achiral, so its solution is optically inactive. Glycine is achiral because its alpha carbon carries two hydrogen atoms. D-glucose and L-proline are chiral, so their solutions rotate plane-polarised light at useful concentrations.

All amino acids except glycine have a chiral alpha carbon. A second chiral carbon must come from the side chain. Threonine has a chiral beta carbon bearing an OH, a CH_3, an H and the alpha carbon, so L-threonine has two chiral carbons. Isoleucine has a chiral beta carbon in its sec-butyl side chain, so it also has two. In contrast, the side chains of leucine (-\text{CH}_2\text{CH(CH}_3)_2) and asparagine (-\text{CH}_2\text{CONH}_2) contain no stereogenic carbon.

• chiral carbon — a carbon bonded to four different substituents.
• n stereocentres — a molecule with n independent stereocentres has up to 2^n stereoisomers, so isoleucine has four.
• achiral — superimposable on its mirror image; such substances are optically inactive.

Mitochondrial respiration inhibitors

Each inhibitor of oxidative phosphorylation acts at a specific point. Cyanide binds the heme a_3–Cu_B centre of cytochrome c oxidase (Complex IV) and blocks electron transfer to O_2. FCCP is a protonophore that carries protons across the inner mitochondrial membrane, dissipating the proton gradient and uncoupling respiration from ATP synthesis. Oligomycin blocks the F_0 proton channel of ATP synthase. Butyl malonate is a competitive inhibitor of succinate dehydrogenase, so it blocks succinate oxidation.

The ATP–ADP translocase (adenine nucleotide translocase) exchanges matrix ATP for cytosolic ADP across the inner membrane. Atractyloside binds the translocase from the cytosolic side and locks it in the c-state, while bongkrekic acid binds from the matrix side and locks it in the m-state. Both block nucleotide exchange. Oligomycin acts on ATP synthase rather than the translocase, and amytal inhibits Complex I.

• uncoupler — an agent such as FCCP that abolishes the proton gradient, so respiration proceeds without ATP synthesis.
• protonophore — a lipophilic weak acid that transports protons across a membrane.
• competitive inhibitor — a substrate analogue that binds the active site, as butyl malonate does for succinate dehydrogenase.

Tryptophan-derived signaling molecules

Tryptophan is the precursor of several bioactive signalling molecules. Serotonin (5-hydroxytryptamine) is formed by hydroxylation and decarboxylation of tryptophan and acts as a neurotransmitter and hormone. Melatonin is produced from serotonin by N-acetylation followed by O-methylation, and it regulates circadian rhythm. Indole-3-acetic acid is the principal plant auxin and is biosynthesised from tryptophan. These are chemically distinct from \gamma-aminobutyric acid, which is formed from glutamate by decarboxylation, so GABA is not a tryptophan derivative.